Project Summary Non-invasive techniques for imaging blood flow down to capillary-level resolution are of paramount importance in order to research and diagnose diseases that have vascular etiology or involvement. Current optical imaging techniques are able to achieve this resolution, but most are typically confined to two dimensions, such as laser-speckle and optical intrinsic-signal imaging techniques, while other techniques, like confocal microscopy, has limited imaging depth (<300m). However, a three-dimensional (3D) visualization of vascular blood perfusion deep within microcirculatory beds at capillary-level resolution is often required to reveal the detailed architecture of the perfused microvascular network so that the volumetric rheology and perfusion status of the tissue can be quantified. We propose to develop, optimize, and characterize 3D optical micro-angiography (OMAG), a novel, non-invasive optical imaging method that can be used to assess vascular perfusion at capillary-level resolution, deep within microcirculation beds. OMAG involves shining a near- infrared light onto a living sample and then analyzing the backscattered light using novel algorithms to obtain, in parallel, volumetric microstructural architecture and blood perfusion images. We will develop rigorous mathematical and experimental models of the backscattering signals originating from a tissue sample in order to better understand how OMAG senses blood flow. We will develop OMAG algorithms to quantitatively assess dynamic blood perfusion in tissue. We will utilize a mouse model (under either normal or pathophysiologic conditions) to assist in developing OMAG. We will obtain transcranial images of cerebral blood flow in the mice and will use the data to improve and validate the OMAG system. This mouse model was chosen because it has been found to be a useful tool in quantifying cerebral blood flow in individual vessels and tissues, in order to better understand mechanisms of human cerebrovascular disease and therapeutic interventions. We will manipulate cortical blood flow by inducing acute thrombotic ischemic stroke (ATIS) in the mice. We will correlate the blood flow images obtained by OMAG with the expected outcomes (experimental end points) of the models. We will also verify the usefulness of OMAG for studying experimental stroke and the effects of pharmacological interventions by confirming that thrombolytic enzyme, tissue plasminogen activator accelerates reperfusion and/or prevents thrombosis progression during ATIS in mice. An exciting aspect of this project is that in the preliminary studies, we have consistently achieved high-quality OMAG images of blood perfusion through the cerebrovascular tree down to the capillary level in mice. These images were obtained through the intact skull of the mouse without the need for dye injections, contrast agents, or surgical craniotomy. Using our preliminary OMAG system, we have been able to capture focal cerebral perfusion cessation during experimental ischemic stroke, with subsequent visualization of the spatial progression of cerebrovascular occlusions over time. The immediate outcomes of this research are twofold: a) a new imaging tool that will allow researchers and clinicians to better understand the pathophysiology of tissue perfusion and ischemic tissue injury, and b) specifically, with respect to cortical brain injury and ischemic tissue perfusion important new information will be obtained in a well-established mouse model. Once validated in this way, we anticipate that the OMAG system will be used to considerable advantage in future studies of stroke and other disorders as new questions arise and as new therapeutic strategies are developed. The OMAG system that we intend to develop in the proposed research will be compact, fast, optically stable, and easily implemented and adaptable in both research laboratories and clinical environments. Project Narrative Visualization and quantization of cerebral blood flow in individual vessels and in tissue volumes are critical for biomedical and clinical research that aims to understand mechanisms of cerebrovascular diseases and therapeutic interventions. Optical micro- angiography will become an important enabling tool in this endeavor because it can provide us with the ability to image in real time the localized cerebro-vascular perfusion at capillary level resolution without the need for dye injections, imaging contrast agent and surgical craniotomy. This imaging system is fast, optically stable, compact, cost- effective, and can be easily implemented and adaptable in both the research laboratories and clinical environments.